Effects of increasing tidal volume and end-expiratory lung volume on induced bronchoconstriction in healthy humans

Background Increasing functional residual capacity (FRC) or tidal volume (VT) reduces airway resistance and attenuates the response to bronchoconstrictor stimuli in animals and humans. What is unknown is which one of the above mechanisms is more effective in modulating airway caliber and whether their combination yields additive or synergistic effects. To address this question, we investigated the effects of increased FRC and increased VT in attenuating the bronchoconstriction induced by inhaled methacholine (MCh) in healthy humans. Methods Nineteen healthy volunteers were challenged with a single-dose of MCh and forced oscillation was used to measure inspiratory resistance at 5 and 19 Hz (R5 and R19), their difference (R5-19), and reactance at 5 Hz (X5) during spontaneous breathing and during imposed breathing patterns with increased FRC, or VT, or both. Importantly, in our experimental design we held the product of VT and breathing frequency (BF), i.e, minute ventilation (VE) fixed so as to better isolate the effects of changes in VT alone. Results Tripling VT from baseline FRC significantly attenuated the effects of MCh on R5, R19, R5-19 and X5. Doubling VT while halving BF had insignificant effects. Increasing FRC by either one or two VT significantly attenuated the effects of MCh on R5, R19, R5-19 and X5. Increasing both VT and FRC had additive effects on R5, R19, R5-19 and X5, but the effect of increasing FRC was more consistent than increasing VT thus suggesting larger bronchodilation. When compared at iso-volume, there were no differences among breathing patterns with the exception of when VT was three times larger than during spontaneous breathing. Conclusions These data show that increasing FRC and VT can attenuate induced bronchoconstriction in healthy humans by additive effects that are mainly related to an increase of mean operational lung volume. We suggest that static stretching as with increasing FRC is more effective than tidal stretching at constant VE, possibly through a combination of effects on airway geometry and airway smooth muscle dynamics. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-024-02909-9.


Introduction
Studies in animals and humans have brought clear evidence that increasing the operating lung volume, i.e., the end-expiratory lung volume above normal functional residual capacity (FRC) or the tidal volume (V T ), reduces airway resistance [1,2] and can attenuate [3] or reverse [4] the response to bronchoconstrictor stimuli.These effects of breathing at increased lung volume can be explained by either static or dynamic mechanisms.Since airways and lung parenchyma are interdependent, a static increase of lung volume is associated with an increase of airway caliber by the action of tethering forces opposing both the passive elastic recoil of the airway wall and the active contractile forces of airway smooth muscle.On the other hand, studies invitro have shown that dynamic swings can blunt the response of airway smooth muscle to contractile stimuli by mechanisms that reduce its force generation capacity [5,6], though in bronchial segments this effect was observed only when pressure oscillations were raised to twice of those corresponding to normal V T [7].In vivo, increasing V T [4], or breathing frequency (BF), or both [8] have a bronchodilator effect.
Therefore, it can be expected that increasing FRC or V T, or their combinations, have beneficial effects in counteracting bronchoconstriction in vivo.However, in porcine bronchial segments, static hyper-distension reduced the maximal response to acetylcholine but blunted the relaxant effect of superimposed pressure oscillations of amplitude corresponding to twice the baseline V T [9], raising the possibility that lung hyperinflation may compete with the bronchodilator effects of increasing V T in vivo.In humans, the relative efficacy of physiologically relevant static hyperinflation and increased dynamic swings in countering airway narrowing has not been studied, but it can be hypothesized that they differ, owing to different underlying mechanisms.
To test this hypothesis, we designed the present study to evaluate whether the bronchodilator effect of breathing at increased lung volumes differs depending on whether attained by increasing FRC or V T .Moreover, we investigated whether the bronchodilator effects of increasing FRC and V T were additive.

Subjects
Nineteen healthy volunteers (13 males/6 females) with no history respiratory/cardiovascular diseases participated in the study.No one was obese.Main anthropometric and respiratory functional data are reported in Table 1.Data were collected at Santa Croce and Carle Hospital (Cuneo, Italy), the protocol was approved by the local Ethical Committee, and each subject gave a written informed consent before participation.

Measurements
Spirometry was measured by a mass flowmeter (Sensor-Medics Inc., CA, USA) following the ATS/ERS recommendations [10].Respiratory impedance was measured by a forced oscillation technique (FOT) as previously described [11,12].Briefly, sinusoidal pressure oscillations (5 and 19 Hz; ~ 2 cmH 2 O peak-to-peak) were generated by a 16-cm diameter loudspeaker (model CW161N, Ciare, Italy) mounted in a rigid plastic box and connected in parallel to a mesh pneumotachograph and mouthpiece on one side and to a low-resistance high-inertance tube on the other side.Pressure oscillations were applied at the mouth during tidal breathing, while subjects had their cheeks supported by the hands of an investigator to minimize upper airway shunting.The overall load over the tidal breathing frequency range was 0.98 cm H 2 O•L -1 •s.Airway opening pressure and flow were recorded by piezoresistive transducers (DCXL10DS and DCXL01DS Sensortechnics, Germany, respectively) and sampled at 200 Hz.A 15-L/min bias flow of air generated by an air pump (CMP08, 3A Health Care, Italy) was used to reduce dead space to about 35 ml.Pressure and flow signals were processed by a least-square algorithm [13,14] to calculate respiratory resistance at 5 and 19 Hz (R 5 and R 19 , respectively) and reactance at 5 Hz (X 5 ).Artifacts due to glottis closure or expiratory airflow limitation were avoided by discarding breaths showing any of the following features: i) tidal volume <0.1 L or >2.0 L, ii) difference between measured flow oscillation and ideal sine wave with the same Fourier coefficients >0.2 [15], and iii) ratio of minimum to average X>3.5 [11].The same breaths were used to measure V T , breathing frequency (BF), inspiratory and total time of each breath (T I and T Tot , respectively), and estimate inspiratory drive (V T /T I ), inspiratory duty cycle (T I /T Tot ), and minute ventilation (V E ).

Pre-study day
Subjects attended the laboratory for spirometry and determination of the dose of methacholine (MCh) to be used for the study day.For this purpose, after baseline FOT measurements, MCh chloride dry-powder (Laboratorio Farmaceutico Lofarma, Milan, Italy) was dissolved in distilled water and administered by an ampouledosimeter system (MB3 MEFAR, Brescia, Italy) delivering aerosol particles with a median mass diameter of 1.53-1.61μm,while subjects breathed quietly in a sitting position.The starting dose was of 300 μg followed by doubling doses until R 5 increased by at least 100% from baseline.

Study day
Baseline FOT measurements were taken during 2 min of spontaneous tidal breathing.Then, the subjects were trained to breathe, by using visual feed-back of spirometry tracing, for 2 min with imposed combinations of FRC or V T .Thereafter, each subject inhaled a single dose of MCh equal to the last dose given on the pre-study day and R 5 was measured 2 min later during spontaneous tidal breathing to confirm the persistence of bronchoconstriction.Then, FOT measurements were taken while subjects maintained for 2 min each of the following imposed breathing patterns in randomized order (Fig. 1): A) spontaneous V T from spontaneous FRC, B) near double V T from spontaneous FRC, C) near triple V T from spontaneous FRC, D) spontaneous V T from FRC increased by 1 V T , E) near double V T from FRC increased by 1 V T , and F) spontaneous V T from FRC increased by 2 V T .For each V T increase the subjects were asked to adjust BF to prevent large increments of V E .Before each change of breathing pattern, R 5 was measured during spontaneous tidal breathing to check for the stability of bronchoconstriction.If R 5 was 10% or more lower than initial post-MCh value an additional half dose of MCh was given to restore bronchoconstriction.This happened occasionally in 6 subjects, with no relation to any specific breathing pattern.
At the end of the study, aerosol albuterol was administered to relieve symptoms if any.
Fig. 1 Patterns of breathing before after methacholine (MCh) with tidal volume (V T ) initiated from spontaneous or increased functional residual capacity (FRC).For each condition, respiratory impedance measures were calculated over the 3 mid-quintiles of the whole inspiratory phase (upper panel) or over the 3 mid-quintiles of iso-volume inspiratory portions (lower panel) as shown by the thick lines

Data analysis
For each breathing pattern, R 5 , R 19 , R 5-19 , and X 5 were calculated over the 3 mid-quintiles of the whole inspiratory phase (Fig. 1, upper panel) or over the 3 mid-quintiles of iso-volume inspiratory portions (Fig. 1, lower panel).Differences in R 5 , R 19 , R 5-19 , X 5 , V T , BF, V T /T I , T I /T Tot , and V E between conditions were tested for statistical significance by a one-way repeated-measure analysis of variance (ANOVA) with Holm-Sidak post-hoc test for multiple-comparisons. Values of p<0.05 were considered statistically significant.Data are presented as mean ± standard deviation (SD).

Breathing patterns during the experimental conditions
The spontaneous breathing pattern after MCh (A) did not differ significantly from the spontaneous pattern before methacholine (Table 2).V T and BF changed with the imposed patterns (B-F) as per protocol.Even though great attention was paid to maintain V E as constant as possible among the imposed breathing patterns, it was with patterns C, E, and F that V E slightly but significantly increased than with patterns than A and B. These differences were associated with significant differences in mean inspiratory, V T /T I .Neither V E nor V T /T I were significantly different among breathing patters C, D, E, and F. There were no significant differences in T I /T TOT among all breathing patterns.

Mid-inspiration measures
In general, breathing at increased FRC, increased V T , or both attenuated the changes induced by MCh inhalation on R 5 , R 19 , R 5-19 , and X 5 (Fig. 2 and Supplemental Table 1).Increasing V T from spontaneous FRC was associated with significant reductions of R 5 , R 19 , R 5-19 and less negative X 5 when V T was tripled (pattern C) but not doubled (pattern B) compared to spontaneous breathing (pattern A) V T .Yet, the attenuating effects of pattern C were significantly greater than those of pattern B.
Increasing FRC by either one (pattern D) or two (pattern F) V T with constant spontaneous V T was associated with significant reductions of R 5 and R 19 than pattern A, while R 5-19 was significantly reduced and X 5 less negative with pattern F but not pattern D.
Increasing both V T and FRC (pattern E) was associated with significantly lower R 5 , R 19 , R 5-19 and less negative X 5 than increasing V T alone (pattern B) and significantly lower R 19 than increasing FRC alone (pattern D).
Breathing patterns with the same peak volume, no matter whether achieved by increasing V T or FRC or both (patterns B vs. D and C vs. E and vs. F) showed insignificantly different effects on airway narrowing.
Notably, R 5 (cmH 2 O•L -1 •s) was reduced by 0.57±1.18when V T was doubled (pattern B vs pattern A), by 1.19±0.70when FRC was increased by 1 V T (pattern D vs pattern A), and by 1.84±0.88when both V T and FRC were increased (pattern E vs pattern A).Similarly, R 19 (cmH 2 O•L -1 •s) was reduced by 0.29±0.35when V T was doubled (pattern B vs pattern A), by 0.48±0.46when FRC was increased by 1 V T (pattern D vs pattern A), and by 0.91±0.42when both V T and FRC were increased (pattern E vs pattern A).These results suggest simply additive effects, but the increase of FRC was more potent to mitigate airway narrowing than the increase in V T .

Iso-volume measures
In general, R 5 , R 19 , and R 5-19 were inversely related to the lung volume at which they were measured (Fig. 3 and Supplemental Table 2), while the X 5 values were inconsistently related to lung volumes.
At low iso-volume, R 5 and R 19 , were significantly lower and X 5 was less negative than during spontaneous breathing (pattern A) when V T was tripled (pattern C) but not doubled (pattern B).Yet, the attenuating effects of pattern C on R 5 and X 5 were significantly greater than those of pattern B.
At mid iso-volume, R 5 , R 19 , and R 5-19 did not differ significantly with increments of V T (patterns B and C), or FRC (pattern D), or both (pattern E).However, X 5 was significantly less negative when both FRC and V T were increased (pattern E) than when V T (pattern B) or FRC (pattern D) were increased alone.
At high iso-volume, there were no significant differences with increments of V T (pattern C), or FRC (panel F), or both (panel E).

Discussion
The main findings of the present study in healthy volunteers were that 1) the changes of respiratory impedance induced by inhaled MCh were significantly attenuated by increasing FRC, or V T , or both, 2) increasing FRC had more consistent effects than increasing V T , 3) the effects of increasing FRC and V T were additive, and ) volume-independent effects attributable to tidal stretching were observed only when V T was three times larger than during spontaneous breathing.

Comments on methodology
We used oscillometry because it is the only available method enabling intra-breath measurements of respiratory mechanics over specific portions of lung volume during tidal breathing, but it has two major limitations.First, oscillometry does not directly measure airway resistance but also lung tissue and chest wall resistances.Airway resistance is inversely related to V T whereas lung tissue resistance is inversely related to BF [2].Therefore, it is possible that the effects of increasing V T on airway caliber were counteracted by the effects of decreasing BF on tissue resistance.We think this had no major effect on our results because the attenuation of R 5 , which reflects in large part tissue resistance, was not less than the attenuation of R 19 , which mainly reflect airway resistance.Second, breathing at increased lung volumes requires activation of inspiratory muscles, which increases chest wall elastance [16].Therefore, we cannot exclude that changes in X 5 with different breathing patterns were counteracted by changes in chest wall stiffness.
Although our subjects were asked to maintain V E as constant as possible by decreasing BF when V T was increased, there was a tendency for V E to increase (Table 2), thus likely resulting in an increased alveolar ventilation and airway hypocapnia, mainly when achieved by increasing V T .Hypocapnia has a bronchoconstrictor effect [17], thus possibly counteracting the bronchodilator effects of imposed breathing patterns.We did not measure end-tidal CO 2 , but we believe this had no major impact on our results for two reasons.First, assuming normal anatomical plus instrumental dead space and CO 2 production, we estimated a mean difference in alveolar PCO 2 between patterns C and A to be approximately 7 mmHg, which was reported to have insignificant effects on the respiratory impedance of healthy subjects [18].Second, the differences in V E between any imposed patters were insignificant and differences in alveolar PCO 2 presumably minimal.
Finally, for changes in V T were associated with changes in BF and the ratio T I /T TOT remained constant, the effects of tissue viscoelasticity could not be evaluated.Nevertheless, breathing patterns with low BF would have increased the time for airway smooth muscle relaxation during the inspiratory phase but also for re-shortening during the expiratory phase.

Interpretation of results
The present study was designed on the premises that both lung hyperinflation and increased breathing depth are mechanisms protecting against airway narrowing, but their relative efficacies are unknown.
That increasing lung volume is associated with a proportional increase of airway conductance, i.e., the reciprocal of airway resistance, was first reported in 1958 by Briscoe and Dubois [1] and subsequently confirmed in excised animal [19] and human [20] lungs with relaxed airways.This effect was simply attributed to a geometric change of airways being distended by the static radial traction of the surrounding lung parenchyma.Studies in contracted airway smooth muscle strips have consistently shown that sustained step-changes of length can rapidly attenuate active tension, possibly due to disassembly of the contractile apparatus, followed by a gradual recovery due to length adaptation [20,21].By contrast, in whole bronchial segments a sustained inflationary increase of transmural pressure also caused an immediate reduction in tension, but this was followed by a continuous gradual decrease [22].Airway wall stiffening was proposed to explain the difference between intact bronchi and muscle strips [22,23].In our study, R 5 was stable or decreased between the different breathing patterns, but never increased, which makes the occurrence of length adaptation unlikely.Thus, it is possible that the attenuations of airway narrowing we observed after 2 min of breathing at increased FRC reflected not only geometric changes in airway caliber but also mechanisms opposing both the passive elastic recoil of the airway wall and the active contractile forces of airway smooth muscle.
The inhibitory effect of cycling stretching on airway smooth muscle active force generation has been reported consistently in both isolated muscle strips [5,6] and isolated bronchial segments [7].It is wellestablished in animals [7] and humans [4,24] that the magnitude of the bronchodilator effects of tidal breathing increases with increasing frequency of breathing and with increasing tidal volume.Two independent lines of evidence suggest, further, that the attenuation of smooth muscle contractile force is attributable to changes of V E , which is the product V T x BF, independently of changes of either V T or BF taken individually [24,25].Equivalently, neither the amplitude of tissue cyclic strain nor the cyclic frequency is as important as their product, namely, the amplitude of the tissue strain rate.To assess this phenomenon still further, in this report we used an experimental design in which we held the product V T x BF fixed so as to better isolate the effects of changes in V T alone.This is an important issue in our study, as we see that when V E could not be kept constant (pattern C vs A) the impedance values at low iso-volume were significantly attenuated presumably because of the higher mean inspiratory flow (V T / T I ) causing a faster lung stretching rate rather than the increase in V T itself.
Three theories can be invoked to explain the above findings [26], namely, that stretching of airway smooth muscle causes a plastic rearrangement of the contractile apparatus [6,27,28], or modifies the crossbridge cycling rate and latch bridges formation [5] or causes temporary detachment of attached cross bridges [29].
In an attempt to examine the relative bronchodilator effects of static hyperinflation and dynamic stretching, we measured inspiratory impedance in healthy subjects with MCh-induced bronchoconstriction breathing with different combinations of FRC and V T .As expected, increasing either V T or FRC significantly attenuated the changes induced by MCh on R 5 and R 19 , R 5-19 , suggestive of a generalized increase of airway caliber, but also decreased R 5-19 and made X 5 less negative.To the extent that an increase in R 5-19 and a decrease in X 5 reflect heterogeneous distribution of time constants within the lung periphery [30], the significant improvement of these variables with the increase in FRC and V T (Figs. 2 and 3) suggests that increasing lung volumes no matter how it was achieved made ventilation more homogeneous.While the effects of increasing V T on R 5 and R 19 were significant only when it was threefold the spontaneous V T , the effects of increasing FRC where already significant when it was increased by one V T , suggesting a more consistent effect of increasing static than dynamic tidal stretching.
The effects of increasing both V T and FRC were additive, i.e., the effect of dynamic stretching was not blunted by an increased static stretch.This finding is in apparent contradiction with a study showing that in isolated bronchial segments hyperinflation blunted the effect of pressure oscillations corresponding to twice a normal V T [9] In that study, bronchi were hyperinflated at a transmural pressure of 20 cmH 2 O, where airway compliance is reduced [7] and so are the amplitude of volume oscillation and airway smooth muscle strain.Examining our data in the light of a previous study [31], (Fig. 3), we estimate that the largest end-tidal inspiratory volumes achieved as with patterns C, E and F would have not exceed the values associated with transpulmonary pressures in excess of 20 cm H 2 O. Since bronchial transmural pressure might differ from transpulmonary pressure in the presence of bronchoconstriction [32], we cannot exclude that stress on airway walls increased with the increase of end-inspiratory volume.Therefore, the increments of V T in our study were likely to reflect increments of airway smooth muscle strain but not stress.The latter, however, does not seem to be the major determinant of the decrease in airway smooth muscle contractility with breathing maneuvers [33,34].
The fact that the effects of FRC and V T were simply additive does suggest that lung hyperinflation and tidal swings operated via a similar mechanism, viz.increase of operational lung volume.This interpretation is supported by the lack of differences at iso-volumes among most breathing patterns.The only exceptions were the lower R 5 , R 19 , R 5-19 , and less negative X 5 at low lung volume after triple V T and the less negative X 5 at mid lung volume with breathing patterns with the highest endinspiratory lung volume, i.e., tripling V T (pattern C) and doubling V T from increased FRC (pattern E).These findings are consistent with a study in airway segments showing modest dilator effects with peak-to-peak pressure oscillations of 10 but not 5 cmH 2 O [7].As FOT measurement were taken during the inspiratory phase, these findings possibly reflect volume-independent dynamic effects on airway smooth muscle persisting after the expiratory phase, even when BF and, in turn, expiratory time for renarrowing was the largest (pattern C).
Why was hyperinflation more potent than tidal swings against airway narrowing in the present study is a matter of speculation.Increasing either FRC or V T results in increased mean operational lung volume, which is associates with an increase of airway caliber owing to the tethering force of lung parenchyma opposing the passive elastic recoil of airway walls.However, the mechanisms of static and dynamic stretching on airway smooth muscle active force may be different.One possibility is that in our study the sustained increments of operational lung volume maintained the airway smooth muscle in a condition of reduced force generation capacity by disassembling the contractile apparatus before the occurrence of length adaptation [20,21] or substantial reduction of tethering force due to stress relation of lung parenchyma [35].By contrast, additional time-dependent effects of tidal stretching, e.g., on cross-bridge cycling rate, were possibly obscured by the re-constriction during expiratory phase unless started from very high end-inspiratory volume.Another possible mechanism explaining the larger bronchodilator effects yielded by the increase in FRC rather than V T could be the larger amount of nitric oxide penetrating the airway lumen when narrowing is relieved by distending lung parenchyma [36].
The results of the present study in healthy subjects cannot be directly extrapolated to asthma because the mechanisms regulating airway smooth muscle contractility and heterogeneity of ventilation may differ in disease.Yet, it is known that FRC increases in asthma with the occurrence of expiratory flow limitation [37] and decreases after bronchodilator treatments [38].Moreover, some beneficial effects of continuous positive airway pressure against airway responsiveness have been reported.To what extent hyperinflation can alleviate asthma symptoms remains to be elucidated, considering that above a given threshold it may cause an increase of inspiratory work of breathing [39] and limit the increase in V T [21].
In conclusion, this study provides evidence that both lung hyperinflation and increased tidal stretching yield substantial bronchodilatation in human lungs exposed to a constrictor agent, though the former seems more effective than the latter presumably because of additive effects on airway smooth muscle contractile force and non-contractile airway tissues.

Fig. 2 Fig. 3
Fig. 2 Effects of increasing tidal volume from spontaneous functional residual capacity (patterns A, B, C) (A), increasing functional residual capacity with spontaneous (patterns A, D, F) (B), or both (patterns B, E) (C) on mid-inspiration impedance measures.Effects of patterns achieving the same peak volume (C vs. E and vs. F) on mean-inspiratory impedance measurements (D).V T , tidal volume; FRC, functional residual capacity.R 5 , respiratory resistance at 5 Hz, R 19 , respiratory resistance at 19 Hz; R 5-19 , difference in respiratory resistance between 5 and 19 Hz; X 5 , respiratory reactance at 5 Hz.Columns heights indicate means and error bars standard deviations.*, p<0.005; **, p<0.01; p<0.001

Table 1
Subjects' anthropometric characteristics and baseline lung functional data BMI Body mass index, FEV 1 Forced expiratory volume in 1 s, VC slow inspiratory vital capacity, R 5 Respiratory resistance at 5 Hz, R 19 Respiratory resistance at 19 Hz, R 5-19 difference in respiratory resistance between 5 and 19 Hz; X 5 respiratory reactance at 5 Hz.Data are mean ± SD

Table 2
Patterns of breathing during experimental conditionsV T tidal volume, BF Breathing frequency, V E , minute ventilation, V T /T i mean inspiratory flow, T i / T tot respiratory duty cycle; FRC, functional residual capacity.Data are mean ± SD